PROJECTSUMMARY/ABSTRACT The understanding we now have of the pHLIPs (pH-Low Insertion Peptides) enables the design of novel, pH- sensitive targeting agents that use the acidity of tumor cell surfaces as a biomarker. The work supported by this grant has already yielded a new diagnostic nuclear imaging agent (PET-pHLIP) and a new fluorescent imaging agent for image-guided surgical interventions (ICG-pHLIP), which are advancing to clinical trials at MemorialSloanKetteringCancerCenterin2019. The primary focus of this continuation is to enable targeted intracellular delivery of polar and moderately hydrophobic therapeutic molecules. We propose a systematic approach using representative cargoes from 3 classes of therapeutic molecules that possess different physical, chemical and functional properties: i) a moderately hydrophobic, sparingly cell-permeable, small drug molecule: mertansine;? ii) a moderately polar, cell-impermeable, cyclic, rigid, larger drug: amanitin;? and iii) a large, polar, cell-impermeable drug: calicheamicin. Each of these drugs is currently under development or in use as an antibody-drug conjugate warhead, but there are important limitations to the antibody approaches, including limited biomarker availability, resistance selection, a narrow therapeutic window and limited delivery, often with < 1% of a constructreachingthetumor.Ourapproachisbasedontargetingtumoracidity,especiallycancercellsurface pHthatisageneralparameterwithinandamongdifferenttumors,andindependentoftumorperfusion. We propose to explore variation of pHLIP sequences, introduce pHLIP-cycles and exploit pHLIP-bundles for delivery of these therapeutic cargoes. The pHLIP-cargo constructs will be tested for insertion stability and kinetics, using biophysical methods and computational modelling. Activity will be evaluated in cells, and promising constructs will be assessed in vivo. We will accumulate a parameter database of pHLIP-cargo properties and employ modern bioinformatics algorithms to analyze the entire data set to reveal major design principles. BystudiesofthesetherapeuticagentswithpHLIPs,wehopetofindprinciplesfortargeteddeliveryofarange of other compounds. Should we be successful, the limitations of antibody drug conjugates for therapy will be overcome,andgreaterunderstandingofthemembranebarrierwillalsobeinhand.Ourexpectationistohave acandidateforthetreatmentofbladdercancerasthepracticaloutcomeofthisgrantrenewal.